The development of an in vitro carcinogenicity assay is described, which utilizes cellular- and enzyme-activating systems from human and rat liver to metabolize carcinogenic chemicals and malignantly transformable C3H1OT 1/2 mouse embryo cells to detect carcinogenic intermediates. Initially, three known potent rat liver carcinogens, aflatoxin B1, 2-acetylaminofluorene and dimethylnitrosamine, plus two weak liver carcinogens, ethionine and safrole, will be studied. Benzo(a) pyrene and benz(a)-anthracene will be used as positive and negative controls, respectively. For most cell-mediated transformation studies, the mouse embryo cells will be seeded on primary or secondary cultures of enzymatically dissociated, lethally irradiated liver cells. For the enzyme-mediated transformation experiments, attached or suspended mouse embryo cells will be treated with post-mitochondrial liver preparation supplemented with the necessary cofactors. After treatment with the carcinogen, the C3H1OT 1/2 cells from both the cell-mediated activation systems will be reseeded for measurement of cytotoxicity and transformation. The effects of the chemicals alone and the activating systems alone on the mouse cells will be investigated. The two metabolic activating systems will be compared and the potential of this type of approach for detection of compounds potentially carcinogenic to humans will be evaluated.